A key survival strategy of RNA viruses is their ability to populate a diverse sequence space that creates a 'cloud'of potentially beneficial mutations at the population level, affording the viral quasispecies a greater probability of evolving and adapting to new environments and challenges during infection. One established mechanism relies on high mutation rates of viral RNA replication. It is becoming increasingly clear that an additional mechanism to expand the evolutionary repertoire relies on the exchange of genetic material between RNA viruses, as well as with genes from their hosts. Furthermore, these recombination mechanisms may provide viruses with two advantages: (i) purge their genomes of accumulated deleterious changes and (ii) create or spread beneficial combinations of mutations in an efficient manner. Despite its importance, the mechanism of viral recombination is not well-understood. Genetic experiments have suggested that homologous RNA recombination occurs by dissociation of the RNA-dependent RNA polymerase and nascent RNA strand before replication completes, and the re-association of that nascent strand-polymerase complex with another template. However, this mechanism remains largely untested. We propose to combine genetics, biochemistry and ultra-deep sequencing approaches with classical virology experiments in cell culture and animal models to define the mechanism of viral recombination and determine its role in virus evolution and pathogenesis. The central hypothesis in this application is that RNA recombination plays a critical role in the generation of virus diversity and evolution and is critical for viral fitness and pathogenesis. PUBLIC HEALTH RELEVANCE: Enteroviruses are a subfamily of small, pathogenic, icosahedral viruses. They have been associated with many clinically recognized, life-threatening syndromes, including poliomyelitis and polioencephalitis, respiratory illnesses, aseptic meningitis, pleurodynia, gastroenteritis, hepatitis, and acute hemorrhagic conjunctivitis. There are no known treatments for these diseases and the only vaccine available to protect against these diseases is the poliovirus vaccine. Virus evolution is at the core of virus drug resistant, immunological survey escape and pathogenesis. RNA recombination is one of the two mechanisms to generate diversity to power evolution. Understanding RNA recombination will help us to understand virus diversity, evolution and pathogenesis, and it may allow the development of safe and effective vaccines and antiviral drugs.